In a Micro Total Analysis System (μ-TAS; Laboratory on a chip), which is assumed a means for analysis of the next generation, a series of chemical and biochemical analyses such as extraction of a target component for analysis from a biological sample (extraction step), analyses of the component using a chemical/biochemical reaction (analytical step), as well as subsequent processing for separation (separation step) and detection (detection step), all are conducted on a extremely small analyzer integrated on a chip, several cm to several ten cm on one side. As for the separating methods for this system, the following methods have widely been noticed. Capillary electrophoresis utilizes a difference of the charge of substances in a high electric field, in which a capillary (fine tube), 1 mm or less in the inside diameter, is prepared with a polymer compound, Teflon or silica as a material easily applicable on a substrate by fine processing. Capillary column chromatography utilizes a difference of the interaction between a column carrier and a substance, using a similar capillary.
Among them, the capillary electrophoresis has characteristics that since the capillary surface area is considerably large relative to the capillary inside volume, generation of the Joule heat by application of high voltage is efficiently blocked and it gives higher resolution in a short period of time than the conventional electrophoresis. Therefore, the capillary electrophoresis has been considered as a method suitable for μ-TAS since the separation is allowed in a relatively short length for separation.
Particularly, recent years, a technique for separation, a so-called capillary chip electrophoresis has been develop as one of capillary electrophoretic methods used for μ-TAS, in which a capillary is made on a chip several cm to several ten cm on one side by means of a fine processing technique such as photolithography. [J. Chromatogr. (1992) 593, 253-258, Manz, A. et al., Anal. Chem. (1992) 64, 1926-1932, Harrison, D. J. et al., Anal. Chem. (1994) 66, 3472-3476, Jacobson, S. C. et al., Science (1993) 261, 895-897, Harrison, D. J. et al., Anal. Chem. (1993) 65, 2637-2642, Effenhauser, C. S. et al. and so on]
In the above-mentioned capillary chip electro-phoretic method, however, there is a limitation in the length of capillary for separation, and the length for separation is extremely shortened in comparison with that of conventional capillary electrophoresis. Therefore, it becomes an issue that separability for relatively large molecules such as nucleic acids, polypeptides, proteins, etc., is insufficient, though it is sufficient for low molecular substances greatly influenced by an intramolecular electric charge.
In order to solve this issue, a method for separation using a polymer having molecular sieve effect, for example, hydroxyethylcellulose, polyacrylamide, and the like, as an additive added into the capillary has been developed. [Proc. Natl. Acad. Sci. USA (1994) 91, 11348-11352, Woolly, A. T. and Mathies, R. A., Anal. Chem. (1996) 68, 720-723, Jacobson, S. C. and Ramsey, J. M., Anal. Chem. (1997) 69, 2181-2186, Woolley, A. T., et al., Proc. Natl. Acad. Sci. USA (1994) 91, 11348-11352, Woolley, A. T. and Mathies, R. A., Anal. Chem. (1994) 66, 2949-2953, Effenhauser, C. S., et al., Anal. Chem (1995) 67, 3676-3680, and so on]. In the existing circumstance, however, separation of polypeptides or proteins is still insufficient even according to these methods.
It is also proposed as another method for separating proteins by using an acrylamide polymer having a molecular sieve effect as a capillary-packing agent in the presence of sodium dodecylsulfate [SDS-PAGE: Proc. Natl. Acad. Sci. USA (1999) 96, 5372-5377, Yao, S., et al., and so on]. In this method, however, since the proteins have to be denatured with sodium dodecylsulfate, it is difficult to separate them while keeping their own activities such as specific binding activity.
In order to solve these problems, the following method has been reported (Japanese Patent No. 3,070,418; International Patent Publication No. 512371/1998). A target component for analysis is allowed to react with 2 species of substances, one which has a specific affinity to the target and binds to a charged substance and another which has a specific affinity to the target and binds to a detectable marker, to form a complex comprising these 3 components (substance which has a specific affinity to the target and binds to a charged substance—target component for analysis—substance which has a specific affinity to the target and binds to a detectable marker). The complex is separated from the substance which has a specific affinity to the target and binds to a detectable marker not involved in the formation of the complex, by an electrically (B/F) separating method utilizing the difference of the charge between the charged substance contained in the complex and the substance which has a specific affinity to the target and binds to a detectable marker not involved in the formation of the complex.
In this method, however, it is necessary that the charged substance improving separation ability and the marker for detection each has been always bound to a different type of substance which has a specific affinity to the target. As an additional disadvantage, it is also necessary to adjust the amount of the detectable marker which is bound to a substance capable of specifically binding to the target component, since the charge of the substance capable of specifically binding to the target component and binding to the detectable marker is changed to decrease the separation accuracy and broaden the separation peak.
In view of the above-mentioned state, the invention intends to provide a method for separating a target for measurement utilizing electrophoresis efficiently in high sensitivity and in a short period of time and a method for measuring the target for measurement separated by the method for separation.